ABSTRACT: This study investigated microbial inactivation via surface-active peracids and used electron spin resonance spectroscopy to characterize the active components and free radical formation. Bacillus atrophaeus spores were injected directly into 3 different concentrations of the peracid disinfectant (1.1%, 1.3%, or 1.5%) for various times (5, 10, 15, or 20 s) at 3 different temperatures (50, 60, or 70 °C) to evaluate the sporicidal activity of the disinfectant mixture. Spectroscopy revealed that the combination of hydrogen peroxide, peracetic acid, and octanoic acid were highly effective at forming a complex mixture of sporicidal, free radical intermediates including hydroxyl and superoxide radicals. Individual components of this mixture alone were not as effective as the final combination. This information has practical applications in the food industry for design of effective sanitation and disinfection agents and suggests that kinetic models could be developed to account for both the physical removal and localized inactivation of spores on food-contact surfaces.